1. Field of the Invention
This invention relates generally to novel analogs of farnesol and geranylgeraniol, and more specifically to 3-substituted farnesol and geranylgeraniol analogs, that block the prenylation of proteins in cells.
2. Background of the Prior Art
Proteins are modified by a mevalonate pathway intermediate. There are three different protein prenylation motifs in this pathwayxe2x80x94farnesylation, geranylgeranylation, and bis-geranylgeranylation. The first modification is carried out by an enzyme, protein farnesyl transferase (FTase), which recognizes the CAAX box (where A=aliphatic amino acid and X=Ser or Met) at the carboxyl terminus of the protein substrate and then attaches the farnesyl group from farnesyl diphosphate (FPP) to the free sulfhydryl of the cysteine residue. FIG. 1 is a reaction pathway for protein farnesylation of, illustratively, a Ras protein. The second, closely related enzyme, protein geranylgeranyltransferase I (GGTase I), attaches a geranylgeranyl moiety from geranylgeranyl diphosphate (GGPP) to a cysteine in a similar CAAX box, where leucine (X=Leu) is the carboxyl terminal residue. The third enzyme, GGTase II, attaches two geranylgeranyl residues to two cysteine residues at the carboxyl terminus of Rab proteins.
Once initial studies demonstrated that the key signal transduction protein and oncogene product Ras is farnesylated, FTase became the subject of intense research interest on the basis that inhibitors of this enzyme could block the action of mutant Ras proteins and halt the growth of ras-transformed cells, and therefore would be potential anti-cancer agents. Mutant forms of Ras proteins are involved in about 30% of human cancers. These include pancreatic adenocarcinomas, colon adenocarcinomas, and adenomas; thyroid carcinomas and adenomas; lung adenocarcinomas; myeloid leukemia; and melanomas. Therefore, there is a need in the art for an FTase inhibitor that can block the growth of ras-transformed cells in vivo.
Significant progress has been made in the development of peptide-based FTase inhibitors, and some of these compounds have shown great promise in vivo as potential anti-cancer agents. Merck has reported a peptidomimetic inhibitor that is effective in vivo in a mouse pancreatic carcinoma model. However, the peptide-based inhibitors are complicated molecules that require numerous synthetic steps to prepare. This limits their availability and increases cost. Moreover, the peptidomimetic inhibitors are not able to penetrate cell membranes due to their polarity. Therefore, in order to be administered in vivo, these drugs must be converted to a prodrug ester form. Even in the prodrug form, the in situ (cell culture) and in vivo potency is several orders of magnitude less than the potency of the parent inhibitor and the isolated enzyme. Furthermore, since peptide-based drugs are usually polar, they are susceptible to proteolytic degradation. This prevents the compound, even in its prodrug form, from being given orally, thereby necessitating dosing by i.v. administration.
While the specificity of FTase for its protein substrate has been extensively explored, there have only been limited reports on its specificity for farnesyl pyrophosphate or farnesyl diphosphate (FPP; Compound 10 on FIG. 1). FPP is a biosynthetic intermediate that occupies a key branch point in the mevalonate pathway. The primary route for FPP metabolism is its conversion into squalene by the enzyme squalene synthase. Squalene is then transformed by a series of enzymatic steps to cholesterol. Squalene synthase has attracted significant interest as a potential additional target for cholesterol-lowering agents. FPP is also converted in the cell to other important isoprenoids, such as dolichol and ubiquinone, which are utilized in protein glycosylation and electron transport, respectively. Most recently it has been recognized that FPP plays an additional crucial role in the cell. It is utilized by the enzyme protein farnesyl transferase (FTase) as the source of a farnesyl moiety that is attached to the cysteine sulfhydryl on the ras G proteins and certain other proteins which bear a carboxyl-terminal Cysxe2x80x94AAXxe2x80x94OH sequence. This farnesylation event and the subsequent proteolysis and carboxymethylation modifications serve to increase the hydrophobicity of these proteins, and thus, convert them to peripheral membrane proteins. There is, therefore, a need for FPP-based FTase inhibitors as probes for analyzing the FPP-binding site of FTase.
Novel FPP analogs were synthesized as probes of the FPP-binding site of FTase and characterized by their interaction with recombinant yeast FTase (yFTase). The vinyl analog, 3-vinyl farnesyl diphosphate (3-vFPP; FIG. 1, Compound 10a) was designed as a potential mechanism-based inhibitor, but instead was a poor alternative substrate for yFTase . These results were reported in Gibbs, et al., J. Org. Chem., Vol. 60, pages 7821-7829 (1995). In contrast, a sterically encumbered analog, 3-tert-butyl farnesyl diphosphate (3-tbFPP; FIG. 1, Compound 10b), is an exceptionally poor substrate and a potent competitive inhibitor of this enzyme. See, Mu, et al., J. Org. Chem., Vol. 61, pages 801-8015 (1996).
The results obtained by the various prenyl transferase inhibitors proposed in the art demonstrate unpredictability with respect to inhibition of FTase or geranylgeranylase (GGTase) in vitro and in vivo. There is, thus, a need for prenyl transferase inhibitors that are effective against mammalian FTase, which is the clinically relevant variant of the enzyme, both in vitro for research purposes and in vivo for therapeutic purposes.
It is, therefore, an object of this invention to provide novel prenyl transferase inhibitors that are easier to prepare than other farnesyl analog FTase inhibitors.
It is also an object of this invention to provide novel prenyl transferase inhibitors that exhibit better cellular penetration.
It is another object of this invention to provide novel prenyl transferase inhibitors that are useful as anticancer agents.
It is a still further object of this invention to provide novel prenyl transferase inhibitors that are bioavailable and non-toxic to the host and that are cytotoxic as compared to other FTase inhibitors that are merely cytostatic.
It is yet another object of this invention to provide novel geranylgeranyl transferase inhibitors that are potentially useful as restenosis inhibitors.
The foregoing and other objects are achieved by this invention which comprises, in a composition of matter embodiment, 3-substituted isoprenol analogs that block the prenylation of proteins in cells. In preferred embodiments, the isoprenol analogs are analogs of the alcohols, farnesol and geranylgeraniol, as well as their diphosphate derivatives. The active, diphosphate derivatives of these compounds are potent inhibitors of mammalian protein prenyltransferases. The alcohol precursors efficiently block the growth of anchorage-independent ras-transformed cells.
The farnesol and geranylgeraniol analogs within the contemplation of this invention, include without limitation,
Formula (I): 
wherein Rxe2x80x2 is a C10-C20 saturated or unsaturated alkyl, aryl, heteroaryl, or cycloalkyl which, in some embodiments, include substituents, some of which may contain heteroatoms, such as N, O, S, and F; R is a C2-C10 saturated or unsaturated alkyl, aryl, cycloalkyl, or C6-C10 aromatic or heteroaromatic group which may be substituted; and X is xe2x80x94OH or xe2x80x94P2O7. As used herein, the composition includes enantiomers, stereoisomers, and geometric isomers.
In preferred embodiments, Rxe2x80x2 is geranyl and farnesyl and R is selected from the group vinyl, ethyl, allyl, saturated and unsaturated isomers of propyl, butyl, and pentyl, cyclopropyl, isobutyl, cyclopentyl, phenyl and heterosubstituted moieties, such as fluorophenyl, (trimethylsilyl)methyl, 1-ethoxyvinyl, and 2-furanyl, 2-thiophenyl.
In a preferred embodiment, the 3-substituted farnesol or geranylgeraniol analogs have of the following formulae (II) and (III):
Formula(II): 
Formula(III): 
wherein R is preferably vinyl, allyl, isopropyl, cyclopropyl, isobutyl, cyclopentyl, or phenyl. However, R may comprises other substituted or unsubstituted C2-C10 saturated and unsaturated alkyl, aryl, and cycloalkyl groups, C6-C10 aromatic groups, and heteroaromatic groups, some of which are illustratively shown in FIG. 2B. As stated above, the isoprenyl alcohols are prodrugs for the active diphosphate form where R is methyl.
Illustrative embodiments of the 3-substituted farnesol and geranylgeraniol analogs are shown on FIGS. 2A and 2B. The compounds are identified on FIGS. 2A and 2B by their common name. Those compounds that are specifically referenced in this application are identified by compound number, and if abbreviated herein, the abbreviation.
In particularly preferred embodiments, the 3-substituted compounds are 3-vinyl farnesol, 3-allylfarnesol, 3-isopropylfarnesol, 3-vinyl geranylgeraniol, 3-allylgeranylgeraniol, and 3-isopropylgeranylgeraniol. In the studies reported herein, 3-allylfarnesol inhibits protein farnesylation in situ and 3-vinylfarnesol instead leads to the abnormal prenylation of proteins with the 3-vinylfarnesyl group. In a similar manner, treatment with 3-allylgeranylgeraniol inhibits protein geranylgeranylation while 3-vinylgeranylgeraniol restores protein geranylgeranylation in cells. Furthermore, 3-allylgeranylgeraniol is one of the most potent inhibitors of GGTase I discovered to date. Studies have now confirmed that the 3-allyl and 3-vinyl analogs are potently cytostatic and cytotoxic to human pancreatic cancer cells (HPAC) in situ. In addition, the 3-allyl analogs have been shown to exhibit selective cytostatic effects on a mouse colon 38 tumor cell line at levels where the same amount has no effect on human fibroblasts. Moreover, preliminary animal studies with severe combined immunodeficiency (SCID) mice indicate that these compounds are non-toxic.
As indicated above, the isoprenol derivatives, 3-substituted farnesol or geranylgeraniol, are precursors, or prodrugs, and as such, rely on the target cells to carry out the biological activation. Therefore, the isoprenol derivative are administered as an anticancer agent, specifically as a prodrug for the active diphosphate, to inhibit the respective prenyl transferases in cells. In particular, inhibition of FTase reduces the level of protein farnesylation in a host, and hence reduces the activity of proteins, such as Ras proteins, which require farnesylation for activation. Of course, these compounds can exert a multitude of other beneficial effects, both known and yet to be discovered.
In a method of use embodiment, a method of treating cancer, specifically cancers of the type which are susceptible to treatment by prenyl transferase inhibitors, comprises administering an effective amount of at least one 3-substituted isoprenol derivative. In particularly preferred embodiments, the 3-substituted isoprenol derivative is selected from the group consisting of 3-allyl farnesol, 3-vinyl farnesol, 3-allyl geranylgeraniol, and 3-vinyl geranylgeraniol. These cancers include any cancer having tumors which are associated with abnormal activity of oncogenes in the ras family, including the three mammalian ras genes, H-ras, K-ras, and N-ras. Other ras proteins include those whose DNA coding regions hybridize to the coding regions of known ras genes. Abnormal ras activity is associated with 30-50% of all lung and colorectal carcinomas and up to 95% of pancreatic carcinomas. Of course, the type of cancers susceptible to prenyl transferase inhibitors is not limited to those bearing ras mutations. Protein prenylation is required for the activity of several signal transduction pathways. Thus, the 3-substituted isoprenol derivatives of the present invention may be used to treat cancers bearing mutations in other oncogenes, including but not limited to various growth factor receptor genes, rho genes, and PTPCAAX genes. Other farnesyl transferase inhibitors have proven to be effective at blocking the growth of cancer cells that do not bear a ras mutation, as well as those that do. See, Cox, et al., Biochim. Biophys, Acta, Vol. 1333, pages F51-F71 (1997).
The novel analogs may be administered alone or in conjunction with other drugs. The analogs may be administered in a variety of ways, orally, parenterally, topically, etc. In injectable forms, the analogs may be delivered subcutaneously, intraperitonealy, or intravascularly. Of course, the analogs may be combined with a delivery vehicle and other fillers, excipients, and the like, as are known in the art to form a pharmaceutical dosage form. Illustrative dosage forms include, tablets, capsules, gels, suspensions, emulsions, liposomes, nanoparticles, etc. Oral dosage forms may be encapsulated or coated, as necessary, in any manner which is known in the art, to protect and/or release the active agent at the appropriate point in the gastrointestinal system.
It has been reported that inhibition of protein geranylgeranylation causes superinduction of nitric oxide synthase (NOS-2) by interleukin-1xcex2 in vascular smooth muscle cells and hepatocytes. Finder, et al., J. Biol. Chem., Vol. 272, No. 21, pages 13484-13488 (1997). This indicates that the 3-substituted geranylgeraniol analogs may inhibit restenosis following angioplasty or other surgical intervention, such as catheterization. It is therefore, contemplated within the invention, that the 3-substituted geranylgeraniol derivatives may be administered to a mammal to inhibit restenosis.
It has also been reported that farnesol inhibits Ca+2 signals in arteries and vascular smooth muscle cells and possesses Ca+2 channel blocker properties. Roulle, et al., J. Biol. Chem., Vol. 272, pages 32240-32246, Dec. 19, 1997. Therefore, it is also contemplated that the novel 3-substituted isoprenol derivatives disclosed herein may be administered to a mammal for their vasoactive properties.